Bacteriophage lambda (λ) is a dsDNA, temperate phage, 50 nm wide and about 150 nm long. Lambda can accept inserts and genomic deletions allowing for insertion of up to 15 kb (Chauthaiwale et al. 1992. Microbiol Rev 56:577-91). Finally, lambda is extremely stable under multiple storage conditions, including desiccation, and large-scale production of lambda is rapid and relatively inexpensive making it a versatile option for vaccine administration to low income nations (Jepson and March 2004. Vaccine 22:2413-9).
Lambda has been used in multiple peptide display experiments (Gupta et al. 2003. Advances in Virus Research 60:421-67; Hoess, 2002. Curr Pharm Biotechnol 3:23-8; Mikawa et al. 1996. J Mol Biol 262:21-30; Santi et al. 2000. J Mol Biol 296:497-508; Sternberg and Hoess. 1995. Proc Natl Acad Sci USA 92:1609-13). Lambda has two identified platforms for peptide display. The tail protein, gpV, consists of 32 subunits important for infection of the bacterial host. After the deletion of a nonessential region of the carboxy terminus, protein and peptide fusions can be successfully displayed on gpV in low copy numbers (Hoess, 2002. Curr Pharm Biotechnol 3:23-8). The second lambda platform for peptide display is the coat protein gpD. The gpD capsid protein is 11.4 kDa and it serves to stabilize the phage head after genomic insertion; there are between 405 and 420 copies of gpD per phage, allowing for higher copy numbers of the displayed peptide. Fusions of both the amino and carboxy terminus have been successfully displayed on the coat surface (Mikawa et al. 1996. J Mol Biol 262:21-30) but resolution of the crystal structure for gpD indicated that the carboxy terminus is well suited to peptide display (Yang, F. et al. 2000. Nat Struct Biol 7:230-7).
Sternberg and Hoess described a method for peptide display in lambda phage. Their base phage lacked gpD, but was stable due to a genomic size of 78.5% of wild type. In addition, the phage possessed a temperature sensitive mutation in the gene responsible for repressing the lytic lifecycle. Therefore, the phage could be stably grown as an E. coli lysogen and the lytic phage released when desired. Transformation of the lambda lysogens with a gpD-peptide expression plasmid resulted in gpD complementation of the phage in trans after lytic induction (Sternberg, N., and R. H. Hoess. 1995. Proc Natl Acad Sci USA 92:1609-13). Eguchi et al. adapted this system for gene delivery to mammalian cells (Eguchi, A. et al. 2001. J Biol Chem 276:26204-10). The Eguchi expression plasmids contained the protein transduction domain of HIV-1 Tat fused to the amino terminus of gpD. In addition, luciferase (luc) or green fluorescent protein (GFP) expression cassettes were inserted into a unique EcoRI restriction site of the lambda genome (λ, D1180) to form λ, D1180(gfp) and λ D1180(luc). Transformation and lytic induction of E. coli lysogens resulted in the formation of recombinant lambda displaying Tat-gpD peptide fusions and capable of delivering either GFP or luc for mammalian cell expression. Successful transduction and subsequent gene expression of COS-1 cells was demonstrated by luciferase assay and fluorescence microscopy. Site-specific GFP expression was also observed after injection of mice with 8.5×109 plaque forming units (pfu) of Tat phage (Eguchi, A. et al. 2001. J Biol Chem 276:26204-10).
Phage are inexpensive to produce and purify, are genetically tractable, and have a substantial track record of safe use in humans and research animals in large quantities for the treatment of bacterial infections (Barrow, P. et al. 1998. Clinical and Diagnostic Laboratory Immunology 5:294-8; Barrow, P. A., and J. S. Soothill. 1997. Trends Microbiol 5:268-71; Dubos, R. et al. 1943. J Exp Med 78:161-168; Schoolnik, G. K. et al. 2004. Nature Biotechnology 22:505-6). The use of phage in vaccine delivery has been proposed, but the development of phage-based vaccines has centered on phage display of antigenic peptides linked to filamentous (M13) coat proteins. These vaccines have successfully induced antibody and some cytolytic responses in laboratory animals (Chen, X. et al. 2001. Nat Med 7:1225-31, De Berardinis, P. et al. 1999. Vaccine 17:1434-41; De Berardinis, P. et al. 2000. Nat Biotechnol 18:873-6), but the T-cell response is often weaker than those observed in mammalian viral vectors. Furthermore, these approaches are limited to short antigenic epitopes, due to the constraints on surface display of peptides on filamentous phage.
Display of proteins in a dense, repetitive array results in strong humoral immune responses, as exemplified by the success of virus-like particles (VLPs) as recombinant vaccine platforms (e.g., for human papillomavirus). However, these approaches have so far failed to elicit the desired antibody response.